(a) Field of the Invention
The invention relates to a bioartificial hydrogel composed of a polymer of ubiquitin units cross-linked with a bifunctionnal polyethylene glycol and derivatives thereof, such as polyethylene oxide, in an aqueous solution. The forming polyubiquitin hydrogel can be used as a wound dressing as a biodegradable delivery vehicle for the systemic or topic delivery of bioactive agents. The hydrogel can also be used also as a biosensor of enzymes, for detection of different nucleic or peptidic molecules. It is defined as a detection condition sensitive system. It further relates to an in situ hybridization system.
(b) Description of Prior Art
Techniques have been developed for administering pharmaceuticals through the skin by absorption. Such techniques are accomplished by devices which typically comprise either a pharmaceutical-containing reservoir enclosed by a synthetic membrane through which the pharmaceutical can diffuse at a controlled rate, or a dispersion of a pharmaceutical in a synthetic polymer matrix in which the pharmaceutical can diffuse at a controlled rate. While such delivery devices work for some pharmaceuticals, the rate of release of other pharmaceuticals is not adequate through synthetic polymers. Either the rate of delivery is too slow to provide an effective dosage given the area of the delivery surface, or in some cases, where prolonged delivery of the drug is desired, delivery is too fast so that the device must be replaced within a short period of time. One situation in which it is desirable to have a drug delivered over a prolonged period of time without removal of the delivery device is the case of delivery of drugs at a wound site around a percutaneous medical device.
Moreover, it is desirable, particularly when dealing with delivery of bioactive agents that are natural products, such as growth factors, that the polymeric matrix from which the drug is delivered be tailored for optimal drug delivery rate. It is difficult to do this when the drug to be delivered is a biological macromolecule, such as an enzyme or surface receptor, since specialized binding functionalities with proper charge density, orientation, hydrophobic domains, etc. are not readily synthesized into synthetic polymers to release the biological macromolecule at a desired controlled rate.
U.S. Pat. No. 4,101,380, the specification of which is incorporated herein by reference, discloses a wide variety of reagents useful to activate polyethylene oxide in the object of obtaining a bifunctionalyzed polyethylene oxide or polyalkene oxide. When those reagents are used to cross-link PEG with a gelatin preformed membrane, a cross-linked gelatin-PEG membrane was obtained and was characterized by a high liquid swelling capacity. However, other embodiments described in the patent provided very low yield of protein cross-linking (in the order of about 2%). The patent states that the use of a carbonate derivative of polyethylene oxide is not recommended and not useful. Attempts should be made to obtain cross-linking of the polymer with a protein or enzyme. This is explained as being due to the high pH required for the subsequent cross-linking reaction which could induce denaturation of enzymes or proteins.
U.S. Pat. No. 5,733,563 discloses albumin based hydrogel for making contact lenses, controlled drug release devices, immobilization matrix for enzymes or cells of therapeutic interest as enzyme correction, wound dressing and artificial skin. The hydrogel contains polyethylene glycol cross-linked with albumin from various sources. Meanwhile, the hydrogel of this invention is characterized by the use of albumin, which gives the possibility to produce hydrogel having only one density. Other limitations of this hydrogel are low resistance to temperature and pH variations, high vulnerability to a great number of proteolytic enzymes, and high potential of inducing allergic reactions.
U.S. Pat. No. 4,615,697 discloses the use of a polymer as moisturizer and humectant and as a bioadhesive vehicle for the controlled release of active principles, in the pharmaceutical field. The synthetic polymer is Polycarbophil, a polyacrylic acid cross-linked with divinyl glycol (3,4-dihydroxy-1,5-hexadiene).
U.S. Pat. No. 5,891,558 features biopolymer foams, composite biopolymer foams, biocompatible constructs comprising biopolymer foams and extracellular matrix particulates and methods for making and using these foams and foam compositions. The foams and foam compositions can be used in vitro, for example, for model systems for research, or in vivo. In either case, the foam compositions can be seeded with cells, e.g., mammalian cells, e.g., human cells, of the same type as those of the tissue which the foams or foam compositions is used to repair or reconstruct. However, collagen sponges, gelatin sponges or polyvinyl alcohol sponges lack biological activity typically present in the extracellular matrix environment of cells, and because of their deficiencies, cross-linked collagen sponges induce little regeneration in vivo or serve poorly as histiotypic and organotypic models in vitro.
U.S. Pat. No. 6,039,940 incorporated herein by reference discloses composition and method for treating a wound with an inherently antimicrobial dressing. The dressing is an hydrogel containing from about 15 to 95 percent, and preferably from about 61 to 90 percent, by weight of a cationic quaternary amine acrylate polymer prepared by the polymerization of acryloyloxyethyl (or propyl)-trialkyl (or aryl)-substituted ammonium salts or acrylamidoethyl (or propyl)-trialkyl (or aryl)-substituted ammonium salts. The antimicrobial hydrogels are non-irritating to the wound, absorb wound exudate, and, due to the inherently antimicrobial properties, enhance the sterile environment around the wound.
Also, the application of recombinant DNA techniques is emerging as a powerful tool in the area of molecular diagnostic medicine. For example, the development of DNA and RNA molecular probes for the detection of viral and bacterial genomes and genetic defects in mammalian chromosomes may replace current immunochemical approaches.
Polynucleotide hybridization assays are used as research tools for the detection and identification of unique or specific polynucleotide sequences in samples of complete, fragmented, or mixed nucleic acids. Various hybridization diagnostic techniques have been developed.
The southern blot technique is based on a polynucleotide hybridization technique employing radiolabeled nucleic acid probes. This procedure permits autoradiographic detection of probe/analyte hybrids and identification of the polynucleotide sequence of the analyte. However, the Southern procedure, as well as the other diagnostic procedures employing radiolabeled nucleic acid probes, are very complex, time consuming, and have the additional problems and expenses generally associated with radioactive materials such as disposal and personnel monitoring. Thus, such assays have remained a tool of basic research and are not generally employed in applied or commercial areas such as clinical diagnosis.
Most of the existing methods used to attach a polynucleotide probe to a solid support are non-specific and the number of attachment sites per nucleic acid is difficult to control. It has been found that multiple attachment reduces the degree of freedom of the immobilized nucleic acid. The physical adsorption of single stranded DNA, covalent attachment via diazo-linkage, epoxidation, cyanogen bromide activation and photochemical reactions are associated with the complication of non-specific linkage between the nucleic acids and the solid support.
Canadian Patent No. 1,223,222, which is incorporated herein by reference, discloses an immobilized nucleic acid-containing probe coupled to a solid support in a manner which is site specific, which does not interfere with the ability of the nucleic acid to hybridize and which involves preferably a single chemical covalent linkage per nucleic acid to the solid support. Specifically, the nucleotide is coupled to the nucleic acid employing an enzyme and the nucleotide is chemically modified.
Canadian Patent No. 1,293,937 discloses polynucleotide probe compositions, diagnostic kits, and nonradiometric hybridization assays useful in the detection and identification of at least one target polynucleotide analyte in a physiological sample. There is provided a first polynucleotide probe having a catalyst attached thereto and which is substantially complementary to a first single-stranded region of the analyte and a second polynucleotide probe having an apoluminescer attached thereto and which is substantially complementary to a second single-stranded region of the analyte. The second region is substantially mutually exclusive from the first region, such that upon hybridization of the first and second probes with the analyte, the catalyst and the apoluminescer are close enough to each other to permit the catalyst to act on a substrate to release a transformation radical to convert the apoluminescer to a luminescer.
Current methods for the diagnosis of inherited diseases employ digestion of a prepared DNA sample with restriction enzymes to form short, double-stranded segments, gel electrophoresis to separate these segments according to size, transfer of the separated segments to a thin membrane material, such as nylon, hybridization of the segments of interest with a labeled oligonucleotide (of complementary sequence to the known disease sequence), and detection of the label. The complete procedure requires about 24 hours, is labor-intensive, and is not readily automated. Furthermore, these methods usually employ radioactive labels, with their inherent safety and disposal problems. None of the above-mentioned diagnostic systems discloses a probe that can be treated to be reusable for hybridization. Thus, these systems are for a unique usage.
A significant drawback in the use of hydrogels, however, and one that has substantially hindered the use of hydrogels in drug delivery systems, is that such formulations are generally not biodegradable. Thus, drug delivery devices formulated with hydrogels typically have to be removed after subcutaneous or intramuscular application or cannot be used at all if direct introduction into the blood stream is necessary. Thus, it would be advantageous to use hydrogels that could be degraded after application in the body without causing toxic or other adverse reactions.
In the art mentioned above, there is no mention or suggest that advantageous hydrogels could be obtained by cross-linking of a polyubiquitin or another native protein in an aqueous solution with activated polyethylene oxide. Therefore, it would be highly desirable to be provided with an improved hydrogel that overcomes or minimizes the above-mentioned problems.
It would also be highly desirable to be provided with a biodegradable hydrogel that has significantly enhanced biocompatibility in that (1) blood compatibility is substantially improved, (2) immunogenicity is minimized, and (3) the hydrogel is enzymatically degraded to endogenous, nontoxic compounds.